GORE. Protein Capture Devices. Operating Instructions Contents. Product Description Device Characteristics... 2 General Handling...

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1 GORE Protein Capture Devices Operating Instructions Contents Product Description Device Characteristics General Handling Flow Direction Flow Rate Selection Buffer and Solutions Protection of Purification Device Antibody Purification Protocol Recommended buffers for use in binding and elution protocols Sample preparation Running Conditions Purification with Clean-in-Place protocol Clean-In-Place Storage Shelf life Labeling Serial Use Troubleshooting Ordering information

2 GORE Protein Capture Devices Product Description GORE Protein Capture Devices are intended for affinity purification of antibodies from clarified feed streams in the drug discovery market and production of antibodies for research and development work. The devices contain a membrane bed with immobilized Protein A. The membrane is made of a unique expanded polytetrafluoroethylene membrane device that provides a binding capacity advantage at high flow rates 30 mg/ml at 20 seconds residence time) and improves the speed of purification. In addition, GORE Protein Capture Devices yield highly concentrated protein, which may eliminate the need for a downstream concentration step. These unique membrane devices are offered in 1.0 ml ( 30 mg capacity) and 3.5 ml ( 105 mg capacity) sizes (Figure 1). Figure 1. GORE Protein Capture Devices GORE Protein Capture Device 1.0 ml GORE Protein Capture Device 3.5 ml Intended Use GORE Protein Capture Devices are intended for research use only and should not be used for clinical or diagnostic procedures. Device Characteristics Table 1. GORE Protein Capture Device Characteristics Device Nominal Bed Volume DBC* Recommended Flow Rate 1.0 ml device 1.0 ml ml/min ml device 3.5 ml mg/ml 10.5 ml/min 105 Capacity for IgG at 30 mg/ml DBC ClP Stability Maximum operating pressure limit 0.1 N NaOH 5 bar (0.5 MPa) *Dynamic binding capacity is determined using human polyclonal IgG at 10% breakthrough with a residence time of 20 seconds. In addition, the 1.0 ml devices were tested with the flow restrictor in-line on the liquid chromatography (LC) system. DBC dynamic binding capacity; CIP clean-in-place; IgG immunoglobulin G; NaOH sodium hydroxide; MPa megapascal 2

3 Operating Instructions General Handling Flow Direction The GORE Protein Capture Devices can be operated with flow running in either direction. If pressure develops over time, the flow can be reversed and cleaning can be performed to reduce the operating pressure. Flow Rate Selection The GORE Protein Capture Devices have a dynamic binding capacity (DBC) of 30 mg/ml at a residence time of 20 seconds, thus allowing for higher flow rates than traditional purification technologies. The flow rate is selected based on pressure. For example, at a residence time of 20 seconds, a 1.0 ml device would have a flow rate of 3.0 ml/min, whereas a 3.5 ml device would have a flow rate of 10.5 ml/min. Buffer and Solutions All liquid chromatography solutions including but not limited to water, chemical solutions, and protein solutions should be highly pure, thoroughly dissolved, and filtered through either a 0.45 μm and/or a 0.2 μm filter to remove particulates, solids, and other large-size impurities. Impurities, protein agglomerates, and particles can play a role in the performance of the liquid chromatography unit and the affinity chromatography device. Protection of Purification Device It is recommended to use an inline 0.2 μm guard column to protect the device, especially if the feed stream or target antibody is known to be susceptible to agglomeration and/or particulates. A maximum pressure setting of 0.8 MPa (8 bar) is recommended to avoid potential damage to the device. The user should refer to the Product Safety Sheet for specific handling procedures. Antibody Purification Protocol Recommended buffers for use in binding and elution protocols Table 2. Recommend buffers for antibody purification using GORE Protein Affinity Devices Buffer Name Chemical Composition ph Conductivity (ms/cm) Recommended Usage Phosphate- Buffered Solution 150 mm NaCl 50 mm Phosphate 7.4± ±1.0 Column equilibration Binding equilibration Citrate Buffer 100 mm Citrate 3.4± ±1.0 Elution buffer Sodium Hydroxide 0.1 N NaOH 13.0± ±1.0 Clean-in-Place buffer DI Water H 2 0 N/A N/A Used for all buffers and aqueous solutions Ethanol (200 proof)/ DI Water Solution 20%/80% (v/v) N/A N/A Buffer for long-term storage of the protein affinity device DI = deionized; ms/cm = millisiemens per centimeter; mm = millimolar; N = Normal; N/A = not applicable; NaCl = sodium chloride; NaOH = sodium hydroxide; v/v = volume/volume 3

4 GORE Protein Capture Devices Optimization of binding and elution buffers may be dependent on the feed stream and antibody and will be specific to the customer. Additional buffers that have been tested include phosphate-buffered solution and glycine, citrate, and Tris buffers. Sample preparation The ph of the feed stream should be adjusted based on the properties of the antibody to be purified to ensure maximum binding to the membrane column. In addition, the clarified feed stream should be filtered through a 0.45 μm and/or 0.2 μm filter to reduce debris and particulates. The purity of the incoming sample will influence device performance and purity level of the eluted antibody. Running Conditions Note: Gore devices are shipped in 20% (v/v) ethanol in deionized water. The device should be rinsed with deionized water before initial use. Purification with Clean-in-Place protocol Note: A clean-in-place (CIP) cycle does not need to be performed after each purification cycle. It is recommended to perform a CIP protocol after every three to five cycles, before switching to a new feed or purification protocol, or after the last purification run prior to storage. The recommended flow rate for all CIP steps is 1.0 ml/min. Note: Flow rates for the purification steps provide a residence time of 20 seconds (3.0 ml/min for a 1.0 ml device and 10.5 ml/min for a 3.5 ml device) unless stated otherwise. In addition, a slower residence time may increase binding capacity. The user can refer to the data sheet for comparison data at various residence times. Note: Differences in the feed source or harvesting conditions may affect purification of the antibody. If an optimization of the purification protocol is needed, consider optimizing sample preparation, buffers composition and ph, residence time, washing protocol, and/or elution protocol. 1. Remove the device from the packaging and wash with deionized water using at least five column volumes Note: Reduced flow rates used in this step will prevent pressure spikes and bubble formation. a. Wash with 5.0 ml at a flow rate of 1.0 ml/min for a 1.0 ml device b. Wash with 17.5 ml at a flow rate of 3.5 ml/min for a 3.5 ml device 2. Equilibrate the device with ten column volumes of phosphate-buffered solution 3. Load the device with the feed containing the target antibody Note: Gore verified the continued performance of the device for at least 20 purification/ cleaning cycles by using clarified and filtered extracts harvested from CHO cells a. Determine sample loading by using one of the following recommendations: i. Load the sample according to the DBC provided on the certificate of analysis ii. Load the sample according to the 10% breakthrough determined from an overload cycle performed prior to purification 4

5 Operating Instructions 4. Wash the device with ten column volumes of phosphate-buffered solution to remove loosely bound material Note: Specific wash protocols can be created and/or optimized based on the properties of the antibody being purified and the purity of the feed stream. 5. Elute the antibody from the device with three to six column volumes of citrate buffer using a 100% isocratic elution Note: Gradient elution protocols can be created and optimized for individual antibodies if needed. Note: Eluted fractions can be neutralized with 1 M Tris buffer at ph 9. It is recommended to use between 250 and 300 µl of 1 M Tris buffer (ph 9) to obtain a neutral ph. The elution ph should be optimized to the isoelectric point of the target protein being purified. In addition, the amount of 1 M Tris buffer (ph 9) used for neutralization may vary depending on the isoelectric point of the target protein. 6. Wash the device with five column volumes of phosphate-buffered solution in the reverse direction of loading 7. Perform CIP cycle by running 0.1 N sodium hydroxide for 15 minutes of contact time at a flow rate at 1.0 ml/minute Note: The customer can refer to the CIP protocol section for additional information 8. Wash the device with ten column volumes of phosphate-buffered solution to equilibrate to a neutral ph 9. Rinse the device with five column volumes of deionized water to remove salt from the device 10. Add five column volumes of 20% ethanol (v/v) in deionized water to the device using the flow rate recommended in step 1 (5.0 ml at a flow rate of 1.0 ml/min for the 1.0 ml device and 17.5 ml at a flow rate of 3.5 ml/min for the 3.5 ml device) Note: Steps 9 and 10 can be disregarded if the device is going to be used for multiple runs. These steps are performed after all purification runs are completed for the day and the device is going to be stored. Table 2 shows column volumes used per purification step for the 1.0 ml and 3.5 ml devices at the recommended flow rates. 5

6 GORE Protein Capture Devices Table 3. Example of an Antibody Purification Procedure # Step Buffer Column Volumes Residence Time (seconds) 1.0 ml Device Flow Rate (ml/min) 1 Equilibration DI Water Equilibration Phosphate-Buffered Solution 3 Load Feed Stream Determined by sample concentration 4 Wash Phosphate-Buffered Solution 5 Elute Citrate Buffer Collection of elution peak 1 6 Wash Phosphate-Buffered Solution 7 Clean 0.1 N Sodium Hydroxide 8 Wash Phosphate-Buffered Solution Not Applicable 2 Not Applicable ml Device Flow Rate (ml/min) 1 Collection was started at an absorbance of 100 mau and continued until the absorbance returned to 100 mau 2 CIP with 0.1 N sodium hydroxide for 15 minutes at a flow rate of 1.0 ml/min. The CIP cycle can be performed as needed. DI deionized; mau milli absorbance unit Clean-In-Place A CIP cycle does not need to be performed after each purification cycle. It is recommended to perform a CIP cycle after three to five purification cycles, before switching to a new feed or purification protocol, or on the last run of the column prior to long-term storage. The CIP is performed with 0.1 N sodium hydroxide. Make sure to allow 15 minutes of contact time at a flow rate of 1.0 ml/min. The CIP cycle can be performed as needed. Do not expose the device to prolonged contact with 0.1 N sodium hydroxide as this can irreversibly damage the device. Please follow the recommended contact times. In addition, do not expose the device to greater than 0.1 N sodium hydroxide. Regeneration and/or sanitization steps with 6 M guanidine hydrochloride or 6 M urea have been used in lieu of a CIP cycle. Optimization of this step will be dependent on the feed stream and will be specific to the customer. Storage Store the device in 20% ethanol (v/v) in deionized water at a temperature range of 2 8 C (35 46 F). It is recommended before use after storage to equilibrate device and perform a blank run, including CIP step. 6 Shelf Life Based on accelerated aging, the GORE Protein Capture Device has a shelf life of one year (12 months) at 2 8 C (35 46 F) under recommended storage conditions.

7 Operating Instructions Labeling Each GORE Protein Capture Device is given a unique identifier to enable traceability back to the master lot and raw material components. Each individual device is labeled with part number and lot number. Each Device package is labeled with product name, part number, lot number, serial lot number, manufacture date, and expiration date. Serial Use GORE Protein Capture Devices can be connected in serial fashion to increase capacity when purifying larger amounts of antibody. Troubleshooting Issue Increase in backpressure Recommendation Evaluate cleaning protocol Optimize sanitization cycle Perform one or more CIP cycles as follows: Perform a CIP cycle in the same direction used for loading Check the back pressure after the CIP cycle Reverse flow direction and perform another CIP cycle if back pressure remains high Ensure that the end of the tubing does not extend 2 mm beyond the end of the fitting Leakage at fittings Loss of capacity over time Protein A leaching HCP/other impurities in elution pool Tighten the 10/32" fittings and ensure proper alignment with the threads Verify DBC and load antibody to the recommended loading volumes (70 to 90% of the 10% breakthrough) Ensure device undergoes a sanitization step or CIP cycle after each purification cycle Protein A leaching occurs with many affinity devices; the device is designed to ensure that leaching per cycle during the life of the device (up to 20 cycles) is 100 ppm Can be antibody dependent and may require additional optimization of the purification protocol to meet specific leaching requirements Follow recommendations below: Load the feed or extract Perform an optional wash step using a buffer with high salt concentration (0.5 to 1.0 M NaCl at ph 7.0) Perform the elution in the reverse direction to remove noncovalently-bound contaminates HCP host cell protein Ordering information Part Number Description Quantity PROA ml Device 1/each PROA ml Device 1/each 7

8 Americas W. L. Gore & Associates, Inc. 402 Vieve s Way Elkton, MD USA Phone: Toll-free (US): pharmbio@wlgore.com Europe W. L. Gore & Associates, GmbH Wernher-von-Braun-Strasse Putzbrunn, Germany Phone: Toll free: pharmbio_eu@wlgore.com All technical information and advice given here is based on our previous experiences and/or test results. We give this information to the best of our knowledge, but assume no legal responsibility. Customers are asked to check the suitability and usability of our products in the specific applications, since the performance of the product can only be judged when all necessary operating data is available. Gore s terms and conditions of sales apply to the purchase and sale of the product. GORE and designs are trademarks of W. L. Gore & Associates W. L. Gore & Associates, Inc. PB6576_Rev2_MA_JUN18 P/N